FACTORS AFFECTING UROCYSTIS‘ COLCHICI
INFECTION. 0F ONION"

Thesis for the Degree of Ph. D.
MICHIGAN STATE UNIVERSITY
.WARD CURTIS. STlENSTRA
1970

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This is to certify that the

thesis entitled
FACTORS AFFECTING UROCYSTIS COLCHICI

INFECTION OF ONION
presented by

Ward Curtis STiensTra

has been accepted towards fulfillment
of the requirements for

Ph.D. degree in Botanx and

Plant PaThology

Date April 30,

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ABSTRACT

FACTORS AFFECTING UROCYSTIS COLCHICI
INFECTION OF ONION

BY

Ward C. Stienstra

The relation of Urocystis colchici teliospore

 

density and disease response in Allium 2223 'Downing Yellow
Globe' was studied. Data on disease severity as a function
of the number of spores/g of soil have been treated by the
multiple-infection transformation, the log-probability
transformation, and the log-log transformation. These
transformations produce linear results for only specific
sections of the disease response curve. Disease was shown
to be linearly related to the distance between propagules
over the entire inoculum range tested.

The effects of planting depth, inoculum placement,
and soil temperature were studied. The amount of disease
increased 13-27% for each 5 mm increase in planting depth,
depending on inoculum density. Eighteen percent disease
occurred from inoculum (1,000 spores/g) present in the top

5 mm of soil when seeds were planted 5, 10, or 15 mm below

Ward C. Stienstra

the surface. However, when a 5 mm zone of inoculum (1,000
spores/g) was placed immediately above the seed, 18, 25,
and 67 percent disease occurred. Maximum infection oc-
curred at 20°C. The time required for emergence was short-
ened at 24 and 28°C, and increased at 12 and 16°C, as
compared to 20°C.

Exudation of ninhydrin- and anthrone-positive
materials by onion seedlings was studied. Exudation after
one day of incubation was 5.0 pg anthrone positive (glucose
equivalent) material/seedling and 2.0 ug ninhydrin positive
(glycine equivalent) material/seedling. Amounts decreased
during the next two days to less than 1 ug/seedling for
ninhydrin- and anthrone-positive materials on the fourth
day.

Nutrients present in dormant onion seeds or in
exudate from seeds incubated 48 hours were shown to support
teliospore germination. Sucrose and asparagine did not
support complete spore germination.

Maximum disease response occurred from inoculum
present in the 5 mm zone immediately above the seeds where
early exudation of nutrients would stimulate spore germina-

tion and the length of exposure to inoculum was the longest.

FACTORS AFFECTING UROCYSTIS COLCHICI

 

INFECTION OF ONION

BY

Ward Curtis Stienstra

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Botany and Plant Pathology

1970

ACKNOWLEDGMENTS

I wish to sincerely thank Dr. M. L. Lacy for his
guidance and encouragement during the course of this inves-
tigation and in the preparation of the manuscript.

Thanks are also due to Dr. J. L. Lockwood, Dr. C. L.
SanClemente, Dr. R. P. Scheffer, and Dr. C. J. Pollard for
their critical evaluation of the manuscript.

I am especially grateful to my wife, Myrna, who
encouraged and supported a student-husband and to our
parents: Mr. and Mrs. Henry Stienstra and Mr. and Mrs.

Arie Schuitman for their moral and financial support.

ii

to my wife and son,

Myrna and Curt

LIST OF TABLES . . .

LIST OF FIGURES . . .

INTRODUCTION . . . .
LITERATURE REVIEW . .
MATERIALS AND METHODS

Source of inoculum

Soil types . . . .
Infestation of soil

Growing plants and assessing disease
Planting depth and inoculum placement

TABLE OF

Time of exposure to inoculum

Exudate collection
Exudate analysis .
Spore germination .

RESULTS . . . . . . .

Effect of inoculum density

Effect of temperature .

Effect of planting depth

Effect of inoculum placement

Time of exposure to inoculum
Onion seedling exudation

Spore germination .
DISCUSSION . . . . .

LITERATURE CITED . .

iv

CONTENTS

Page

12
13
13
14
15
16

17
18

O O O O O
H
m

31
34
36
38
42

O O O O O O O O O O O O 51

O I O O O O I O O O O O 59

LIST OF TABLES

Table Page

1. The effects of inoculum density on percent
disease and number of infections at
three planting depths . . . . . . . . . . . . 21

2. The effects of planting depth and infestation
of soil by zones on percent disease . . . . . 35

3. Number of days after planting required for
50% emergence of cotyledons and 50%
appearance of true leaves . . . . . . . . . . 37

4. Germination of Urocystis colchici teliospores
on agar media and on water agar placed over
onion seeds or agar media . . . . . . . . . . 46

 

5. Germination of Urocystis colchici teliospores
on malt extract-phytone agar at 22°C after
different treatments . . . . . .~. . . . . . . 48

 

6. Germination of Urocystis colchici teliospores
on sucrose and asparagine in agar media at
22°C 0 o o o o o o o o o o ' o o o o o o o o o o 50

 

Figure

l.

10.

LIST OF FIGURES

Page

Effect of inoculum density on percent
smut disease . . . . . . . . . . . . . . 23

Effect of inoculum density on the number
of infections . . . . . . . . . . . . . 24

The relation between percent smut disease
(probability scale) and the number of
Spores/g of soil (logarithmic scale) . . 25

The relation between the log number of
infections and the log number of
spores/g of soil . . . . . . . . . . . . 27

The relation between percent smut disease
and the calculated distance between
spores . . . . . . . . . . . . . . . . . 28

The effect of temperature on disease in-
cidence at two inoculum levels . . . . . 30

The effect of relative host surface area
exposed to four inoculum densities on
percent disease . . . . . . . . . . . . 32

Conductivity of exudates of onion seeds
germinating under moist glass fiber
filter paper and leached daily . . . . . 39

Carbohydrates and amino acids exuded by
onion seeds germinating under moist
glass fiber filter paper and washed
daily . . . . . . . . . . . . . . . . . 41

Carbohydrates exuded by uncontaminated
onion seedlings after seeds were in-
cubated four days (X) on agar and
then placed in sterile chambers and
washed daily . . . . . . . . . . . . . . 43

vi

Figure Page

11. Amino acids exuded by uncontaminated
onion seedlings after seeds were
incubated four days (X) on agar and
then placed in sterile chambers and
washed daily . . . . . . . . . . . . . . 44

12. The relation between calculated distance
(D) between spores in soil and number
of spores or log number of spores/g
of soil . . . . . . . . . . . . . . . . 53

vii

INTRODUCTION

Smut fungi played an important role in the early
history of plant pathology. Tilletia caries, incitant of
stinking smut or bunt of wheat, was used by Tillet (43) in
1755 to establish the principle that plant diseases were
contagious. Prevost (31) in 1807 observed germination of
wheat bunt spores and conceived the idea that this fungus
penetrated the wheat plant and caused disease. The revolu-
tionary discovery that fungal spores germinated as "seeds"
of microscoPic plants and produced new individuals was not
accepted until 1847, when published studies on the wheat
bunt fungus confirmed Prevost's observations of spore ger-
mination and fungal growth in the host plant (45). These
early studies on smut diseases served as models for later
scientific work on the nature and causes of plant disease.

The onion smut fungus (Urocystis colchici (Shlecht.)

 

Rabenh.) is a soil-borne plant pathogen (l, 6, 42, 45).
The overwintering stage is the teliospore, often described
as a chlamydospore. The thick-walled teliospore is sur-
rounded by 15-20 small sterile cells and is reported to
contain a single diploid nucleus (1, 6, 13, 42, 45).

Tachibana and Duran suggested that g. colchici teliospores

exhibited bipolar sexuality and produced unisexual mycelia
(41). The nucleus is believed to undergo meiosis prior to
teliospore germination (6), but Tachibana and Duran (41)
were unable to infect onion seedlings with mycelium from
single spores.. The germinating teliospore produces a
branching monokaryotic promycelium from which hyphae develop
which directly penetrate the epidermal cells of the onion
cotyledon (13). The mycelium progresses intercellularly,
with the host cells being pushed apart as the lesion grows.
Systemic infection is associated with the penetration of
the mycelium to the onion meristem. The mycelium becomes
dikaryotic just prior to teliospore formation (6), which
can occur in the cotyledon, bulb scales, or leaves.

Smut was one of the first groups of diseases to be
controlled by chemicals (24). Seed treatments collectively
known as lg_Chaulage, involving lye, ammonia, or copper
sulfate were used for the control of stinking smut by
European farmers in the late 1700's. Chemical control
methods for onion smut were developed in the early 19th
century (42). The earliest control was a solution of
formaldehyde dripped in the furrow near the seed (35).
Formaldehyde inhibited the fungus until the onion plant
develOped beyond the three-week susceptible period. Smut
control now consists of either seed treatment or in-furrow
soil fumigation. This disease cannot be economically

eradicated from soil because of longevity of the fungal

spores, and therefore remains an important disease in the
northern onion growing areas despite effective control
measures (45). Environmental conditions affect the period
of susceptibility and the amount of infection (46). Infec-
tion is limited in climates with mean soil temperatures
exceeding 25°C at the time of planting, apparently because
of inhibition of the pathogen (12, 23, 47). In addition
the onion seedling develops rapidly under these conditions,
shortening the period of susceptibility (46).

The purpose of my research was to study the factors

affecting Urocystis colchici infection of onion. These

 

factors included inoculum density and placement, planting
depth, temperature, and host exudates. Inoculum density
has a profound influence on disease severity; therefore,
most of the research reported herein was directed toward

evaluating the relation of inoculum density and disease.

LITERATURE REVIEW

The challenge of dealing in a quantitative way with
soil-borne inoculum was summarized by Dimond and Horsfall
(11). They state that air-borne pathogen concentrations
can be estimated by spore trapping, but in soil estimation
is very difficult. Air—borne inoculum can be applied
directly to the host surface and measurements made of the
relationship between inoculum density and resultant disease
production, but in soil direct observations are more diffi-
cult because of the opacity of soil. Data from diseases
with air-borne inoculum have given reasonably satisfactory
theories for predicting distribution of inoculum and disease
severity, but in soil this has proven less applicable be-
cause of the relative immobility of inoculum and difficulty
of direct observation of infection of host plants.

The various definitions of the term "inoculum
potential" in soil have been reviewed recently by Dimond
and Horsfall (10), Garrett (15» and Baker (3). Inoculum
potential has been defined as the energy available for
infection at the surface of the host to be infected (14).
This is a resultant of the inoculum density and the envi-

ronmental or capacity factors affecting the inoculum.

Disease potential has been defined by Grainger as the
ability of the host to contract disease at different stages
in its development (16). I will use the formula developed
at the discussion following the symposium on Ecology of
Soil-borne Plant Pathogens held at Berkeley in which:

Disease Severity = Inoculum Potential x Disease
Potential.

Data of Rowell and Olien (32) indicated a simple,
linear relation between the amount of inoculum and number
of infections when the amount of inoculum was small. How-
ever, as inoculum became more abundant the resultant number
of infections was no longer simply related to the number of
diseased plants because a plant could be infected a second
or third time and still be counted as one infection.
Disease measured as percent diseased plants thus ignores
the increase in the number of infections that occur on
diseased plants (17). This shortcoming of assessing disease
by percentage diseased plants led to computation of the
number of infections corresponding to different percentages
of diseased plants by Gregory (17). This multiple-infection
transformation was called the semilogarithmic transformation
by Dimond and Horsfall (11). A similar transformation
prOposed by van der Plank (30) was loge l/l - X, where X =
the percentage of diseased plants and l = unity, or all
susceptible plants in the population. His argument was

that new infections occurred only in tissue not previously

infected. Gregory and van der Plank thus arrived at the
same result from two different approaches. Gregory cor-
rected for multiple infections by computing the number of
infections corresponding to different percentages of
diseased plants, while van der Plank assumed that multiple
infections did occur and dealt with the number of healthy
plants remaining.

Another hypothesis which deals with the relation
between inoculum and disease states that the logarithm of
inoculum density is linearly related to the probability of
a host becoming infected (19, 49). Since living organisms
are variable, it can be assumed that in a population of
inoculum, virulence is not uniform, and in a population of
infection sites, susceptibility is not uniform (11). The
probability of highly virulent spores infecting less sus-
ceptible infection sites and the probability of less vir-
ulent spores infecting highly susceptible infection sites
is smaller than the probability of average sites being
infected by average spores. That is, virulence in the
pathogen and susceptibility in the host follow a normal
distribution. Therefore the response scale needs to be
stretched at both ends to provide equal information for
equal distance on the scale (19). The probability scale
was employed for this purpose. The application of the log-
probit grid was shown to hold for inoculum levels, using

the data of Heald (18) on spore load of hunt in relation to

the percentage of smutted wheat plants, as well as for
response of fungal spores to fungicide dosage (9). However,
log-probit dosage-response curves become flat at high
inoculum densities because multiple infections are ignored
(11). Furthermore, this analysis can only straighten a
symmetrical S-shaped curve.

The approaches described above have been used to
straighten dosage—response curves so that the relation
between disease and inoculum was linear. Pathologists
interested in soil-borne inoculum have long attempted to
define the events affecting the process of infection in
soil. Since plants merely influence pathogens in soil by
growing into proximity of pathogen prOpagules, infection
is a matter of probability of contact of host and pathogen
and is directly related to the number of spores that are in
a position to be influenced. Therefore the geometric
orientation of spores in soil should be examined.

Baker (2, 3) prOposed that orientation of spores in
soil could be as a lattice of trahedra, since the tetra-
hedron was the simplest possible three-dimensional figure
conceivable. This hypothesis requires an ideal situation
in which any addition of inoculum to a soil system redis-
tributes all inoculum, so that the propagules initially
present and the added propagules always reorient themselves
to become equidistant from each other. In cultivated soils

the actual situation may approach the ideal (3). The

tetrahedral surface consists of four equilateral triangles,
and each apex is equidistant from the other three. If each
apex represents a spore, the distance between spores, or
length of an edge of the tetrahedron, is directly propor-
tional to the volume enclosed within the tetraderon. ‘The

volume of a tetrahedron (Vt) is described by the formula:

vt = 0.11785 D3 (I)

where (D) is the distance between apices, or spores. While
tetrahedra do not completely "fill Space," other geometric
figures are less suitable (3).

In soil the influence of roots on propagules is one
of two basic types: a rhizosphere or rhizoplane effect (2,
3). In the rhizosphere type, a cylinder of soil adjacent
to, but not touching the root is affected; that is, the
host root, hypocotyl, epicotyl, or cotyledon is surrounded
by a cylindrical volume of soil under the host's influence.
If pathogen propagules germinate and grow in the rhizosphere,
Baker (3) stated that any additional inoculum in the soil
volume influenced by the host should produce a directly
prOportional increase in disease. From equation (I) the
number of points in a unit volume is inversely proportional

to the cube of the distance between them:

I = 3 (II)

where (I) is inoculum density (spores/g), and (k) is a
constant. The number of infections (S) as a result of
rhizosphere influence is also inversely proportional to the

cube of the distance between spores:

(III)

UIW
U)

The number of infections (S) can be expressed in terms of

the number of spores/g as follows:

S = I (IV)

Thus the number of infections is directly prOportional to
the number of spores/g of soil, if the zone of influence is
a rhizosphere (2, 3).

However, in the rhizoplane type, only propagules
touching the host surface infect, and theoretically no
volume of soil is influenced (3). The host root, hypocotyl,
epicotyl, or cotyledon surface area is the infection court,
and only pathogen propagules in direct contact could ger-
minate and infect. Thus, when inoculum is added to soil,
it is essential to determine what proportion would be in
contact with the host surface. The number of points of a
tetrahedral lattice touching a unit surface area is two
dimensional and inversely proportional to the square of the

distance between the points:

(V)

UIW
N

10

The number of infections (S) can then be expressed in terms

of (I). The 2/3 power of equation (II) is:

2/3 k2/3

I - -37—- (VI)

which can be rewirtten as:

D = i77§' (VII)

Equation (VII) can be substituted into (V) as follows:

k 2/3

S = = kI (VIII)

 

The slope of the curve resulting when Log S is plotted
against Log I can be determined from equation (VIII) when

it is written in log form:

log S = 2/3 log I + log k (IX)

The slope of log S plotted against log I is 2/3.

Thus, Baker (3) hypothesized that the slope of the
curve resulting when log number of infections is plotted
against log inoculum density should be 2/3 for a rhiZOplane
influence and one for a rhizosphere influence. Very little
data are available to test the hypothesis.

Exudation of soluble organic substances is a phenom-
enon common to all higher plants (38). The importance of

exudates in influencing soil microorganisms, both pathogens

11

and saprophytes, has been demonstrated for many plants.
Major sources of exudates are: the hilum-micropylar area
of intact germinating seeds (29, 36), the root, the region
of developing root hairs, and sites where breaks appear in
the eipdermis (29, 32, 39). The types of substances found
in exudates are materials involved in cellular metabolism.
Soil-borne plant pathogenic fungi typically invade only
living tissue and do not grow through nonsterile soil.

They generally exist in soil apart from the host as resting
spores or sclerotia. Infection is promoted by the growth
of the host into the micro-environment of the dormant spore.
Thus as the plant invades the fungus habitat, it alters the
micro-site nutritionally to favor spore germination and
growth. There is good evidence that many propagules in
soil require a carbon and nitrogen source for germination
(7), and this stimulus is provided by the exudates from
host plants (5, 8, 20, 28, 34). Spore germination in the
rhizosphere or rhiZOplane is not selective for pathogens
(37, 44), but it appears to be a general response of many
microorganisms to nutrients exuded from plant parts (40,
25). If, however, exudates from a suitable host induce
spore germination of a pathogen, the possibility of a suc-
cessful infection exists. Kinds and amounts of exudates
profoundly influenced amount of infection resulting from
inoculum by influencing germination of pathogen propagules

(20).

MATERIALS AND METHODS

Source of inoculum

 

Onion smut teliospores were collected in June of
each year from naturally infected field-grown onion plants
with at least one visible sorus per plant. The infected
areas were removed and allowed to dry at room temperature.
The dry leaf sections were fragmented in a Waring blender
and sieved to remove most of the plant material. Material
passed through a 200 mesh sieve contained approximately 2
x 108 teliospores/g. Microscopic examination indicated a
high percentage of smut spores with only an occasional spore

of Helminthosporium or Alternaria. This material was stored

 

 

in a stoppered vial at 2°C, and used as inoculum for soil
infestation.
Field-collected spores were used as inoculum because

Urocystis does not sporulate in culture. It was impossible

 

to assay the viability of inoculum directly because contam-
inating microorganisms quickly overrun the slowly germinating
smut spores (l). The viability of inoculum was assayed
indirectly by determining the amount of inoculum required

to give 50i5% infection under optimum conditions with

freshly collected inoculum. If inoculum later fell below

12

13

this level of disease incidence more than 10%, the exper-

iment was repeated with a newer inoculum collection.

Soil types

 

Since virtually all Michigan onions are grown on
organic soils, it was first thought that this type of soil
would be used for soil infestation experiments. However
organic soils are difficult to handle because of their
physical properties. A comparison of disease response to
several inoculum levels was made using organic soil, a
mixture of organic soil and fine sand (3:1 V/v), and the
Michigan State University greenhouse mixture of Conover
loam, sand, and peat (1:1:1 v/v). The results indicated
that no difference in disease incidence existed between
soils, and the pasteurized greenhouse mixture was used in

all further studies.

Infestation of soil

 

Soil was infested at varying inoculum densities by
mixing a known number of teliospores into a known weight of
soil corrected to an oven dry basis. Soil and spores were
mixed for 20 to 30 minutes in a concrete mixer. Infested
soil was diluted by mixing with uninfested soil to obtain
lower inoculum densities. Later, estimates of soil bulk
density were made, which allowed calculation of the mean

distance between spores (2, 3).

14

Growing plants and assessing
disease

Surface-disinfested Allium 2223 L. 'Downing Yellow
Globe' seeds were planted either in eight inch plastic pots
or metal flats (64 x 39 x 10 cm). To insure uniform plant-
ing depth in pots, seeds were pushed into soil with a small
rod to a selected depth. Seeds planted in metal flats were
placed on the smooth surface of a compact soil. They were
then carefully covered with more soil, which was smoothed
and compacted until the seeds were at the desired planting
depth. The pots or flats were either placed in water bath
controlled temperature tanks, growth chambers or on green—
house benches, depending on the experiment, and fertilized
weekly. The greenhouse was cooled by an evaporative cooler,
and experiments have not been reported in which summer
temperatures (above 25°C) may have interfered with the
infection process. Natural light was supplemented with 16
hours of artificial light from four 40 watt fluorescent
bulbs (1/2 warm and 1/2 cool white) suspended 18 inches
above the bench for each 16 square feet of space. A minimum
of 100 seedlings in each of four replicate pots were observed
for smut infection. The criterion used for determining
infection was visual perception of sori. All experiments
were repeated at least twice with similar results. Varia-
bility in any given inoculum density experiment did not

exceed i5% diseased plants.

15

Planting depth and inoculum
placement

Initial planting depth studies were carried out in
eight inch plastic pots with an internal diameter of 19 cm.
The pot was filled with a four inch gravel base and infested
soil was added, leveled, and compacted. Seeds were placed
on the smooth soil surface and covered with a measured
volume of infested soil calculated to position the seeds
10 mm below the soil surface. Seeds could be planted at
selected depths more rapidly and uniformly using the volume
method than trying to prepare furrows or push seeds into
soil individually. Later, to study the effect of inoculum
placed in zones, another method of planting became neces-
sary. The rectangular metal flats were used and a straight
edge which could be adjusted to selected depths in the flat
was used to level the soil. Soil was built up and compacted
to a desired depth. Seeds were placed on the smooth, level
soil surface and carefully covered with more soil. The
straight edge was adjusted 5 mm above the seed placement
depth and the newly added soil was leveled, compacted, and
built up to the selected height. This process was repeated
using infested soil or uninfested soil. This method allowed
for precise control of planting depth and soil could be

infested in layers by increments of 5 mm.

16

Time of exposure to inoculum

Length of exposure to inoculum may be important in
determining disease response. Longer exposure of suscep-
tible host tissue to inoculum should result in increased
amounts of disease (3). The effect of planting depth and
temperature on the length of time plants were exposed to
inoculum was measured. Counts were made daily on cotyledon

emergence and appearance of the first true leaf.

Exudate collection

 

Onion seeds were surface disinfested by immersion in
a 10% Clorox solution for 10 minutes. The seeds were rinsed
with sterile distilled water and treated in one of two ways.

(i) Four 9 of seeds were placed in a Buchner funnel
and covered with a glass fiber filter paper. Distilled
water slowly dripping onto the filter paper was collected
either by suction through a 0.45 p Millipore filter, or in
a sufficient volume of ethanol to insure a final concentra-
tion of not less than 50% ethanol after 24 hours. Ethanol
or filtration was used to prevent utilization of exudates
by contaminating organisms.

(ii) Seeds were placed on a medium containing 5
g/liter each of yeast extract-malt extract-peptone (YEMEP
medium) and assayed for sterility. Twenty five to 35 seeds
and seedlings that remained sterile after four days were

placed onto petri dishes 7 cm deep containing a base layer

17

of acid-washed, sterile fine sand and covered with a 1/2
inch layer of acid washed, sterile coarse sand. The dishes
were specially modified with an inlet in the cover and an
outlet in the side near the base (22). At 24 hour inter-
vals, 100 ml of sterile distilled water was applied to the
surface of the sand and collected in a sterile flask. All
solutions containing exudates were tested for sterility on
YEMEP medium. The exudate—containing solutions were reduced
from 100 to 10 ml under vacuum in a rotary evaporator at

40°C and frozen until analyzed.

Exudate analysis

 

Conductivity of the exudate solutions was determined
with a conductivity bridge. Results are expressed in u
mhos/seed and are corrected for background conductance.

Total carbohydrates were determined using a modifi—
cation of Dreywood's anthrone reagent (27). The anthrone
reagent was prepared by dissolving 0.2 g anthrone in 100 ml
of 95% H2 SO4 (one liter concentrated sulfuric acid and
50 ml of water). One ml of the solution to be assayed was
mixed with 9 ml anthrone reagent and placed in a boiling
water bath for ten minutes. The solutions were cooled and
the optical density at 600 mu was determined with a Bausch

and Lomb Spectronic 20 spectrophotometer. A standard curve

was made using glucose (20-100 ug/ml).

18

Total amino acids were determined by the method of
Moore and Stein (25). Ninhydrin reagent was made by dis-
solving 0.2 g ninhydrin and 0.03 g hydrindantin in 7.5 ml
ethylene glycol monoethyl ether and 2.5 ml 4 N sodium
acetate: acetic acid buffer (pH 5.5). The buffer was made
0 to 40.0 ml H

by adding 54.4 g sodium acetate - 3H 0,

2 2
mixing in 10 ml glacial acetic acid and diluting to 100 ml
with water. One ml of the sample to be assayed was mixed
with one m1 ninhydrin reagent in a boiling water bath for
15 minutes. The solution was diluted with 8 ml 50% ethyl
alcohol, and optical density determined at 570 mu in a

spectrophotometer. A standard curve was made using glycine

(4-32 ug/ml).

Sporeggermination

 

Urocystis colchici teliospores for germination
studies were collected from 6-12 week-old onion seedlings
grown in greenhouse soil infested with field-collected
teliospores (23). Plants with sori in the leaves were
immersed for ten minutes in 10% Clorox (containing one drop
of Tween 20 surfactant/liter) to disinfest the leaf surface.
The plants were rinsed three times in sterile distilled
water and allowed to dry in a transfer hood for 10-15 min-
utes. Intact leaves containing sori were slit with a
sterile spatula, the spores scraped from the lacunae of the

leaves and suSpended in sterile distilled water at a

19

concentration of 105 - 106 spores/m1. Single-spore units

for germination studies were obtained by breaking spore
clumps with high speed agitation in a sterile Sorval Omni-
mixer. The spore suspensions were stored at 20-22°C for
24 hours and diluted to 2 x 103 spores/m1. One quarter ml
of the suspension was spread evenly over the surface of
each of four replicate agar plates with a sterile glass
rod. Spores plated at this concentration were sufficiently
separated so that colonies developing from germinating
spores did not overgrow and conceal ungerminated spores.
All agar media used in spore germination studies
contained (g/liter): KH2P04, 4,
FeSO4, 0.01; and bacteriological agar, 15.0. Onion seeds,

1.0; KCl, 0.5; MgSO 0.5;
broken with high speed agitation in a Sorval Omni-mixer,
were used to amend agar, and intact seeds, autoclaved or
surface disinfested with 10% Clorox for 10 minutes, were
placed under solidified water agar to determine the effect
on spore germination. All media were adjusted to pH 6.2
and amount of germination was determined after eight days
at 22°C. One hundred spores were observed for germination
on each of four replicate plates. Spores were considered
germinated if germ tubes were as long as spore diameter.
Spore germination level and rate were compared to the level
and rate obtained on malt extract (10 g/liter)-phytone (5
g/liter) medium reported to be optimum for teliospore ger-

mination (23). All experiments were repeated at least twice.

RESULTS

Effect of inoculum density

 

The effects of inoculum densities (125 to 8,000
spores/g of soil) on disease incidence when measured by
percent diseased plants (X), and on disease severity by
the calculated number of infections (S), are presented
(Table 1). Also presented are the calculated mean distances
between spores as inoculum density is increased, calculated
assuming that spores in soil are distributed uniformly in a
three dimensional medium (2, 3). The number of spores
required to establish a selected distance between spores in
a given volume of soil, assuming spores represent the apices

of tetrahedra, is calculated by the formula:

V
N= 5 3 (x)
(0.11785 D )

 

where N is the number of spores per volume of soil, V8 is
the volume of soil to be infested, and D is the distance
between spores desired.

Percent diseased plants and calculated number of
infections increased with increasing inoculum density and

with decreasing distance between spores. Amount of disease

20

21

Table l.--The effects of inoculum density on percent dis-

ease and number of infections at three planting

 

 

 

depths
Number
Spores/g D(mm) Planting depth
10 mm 15 mm
X S X S X S
125 4.1 1 1 22 25 34 42
250 3.2 15 16 29 34 46 62
500 2.6 20 22 36 45 63 99
1,000 2.0 35 43 56 82 82 172
2,000 1.7 49 67 62 97 89 221
4,000 1.3 44 58 67 111 91 241
8,000 1.0 56 82 77 148 92 253

 

distance between spores in mm

percent infection

number of infections per plant

22

at any inoculum density also increased with deeper planting.
These data were analyzed several ways in Figures l-5.

Percent diseased plants typically increased very
rapidly at first and then the curve flattened as inoculum
density was further increased (Figure 1). An arithmetic
plot of this type produces a curve that is very difficult
to analyze, since most of the increase in disease response
occurs in a small inoculum range, and additional inoculum
appears less effective in producing a unit disease response.
There is not a simple relation between the number of infec-
tions and the percent diseased plants because a plant can
become infected a second or third time and still be counted
as a single diseased plant. The data were therefore plotted
as the calculated number of infections versus the number of
spores/g of soil (Figure 2). In contrast with Figure l
where multiple infections are ignored, Figure 2 corrects
for the increase in number of infections as inoculum density
increases. These curves, while more nearly linear than the
curves in Figure l, were difficult to analyze because the
response to increasing inoculum was not constant.

A logarithmic-probability plot (Figure 3) usually
gives a linear relation between the percentage of disease
units on a probability scale and the amount of inoculum on
a logarithmic scale (ll, 19). This hypothesis assumes that
susceptible plants are normally distributed in accordance

with the logarithm of the inoculum level. Therefore the

23

.ommomHU ucEm ucmoHom co muflmcoc Edasoocfl mo pomwmm

Hwom m\mmuomm

.H musmflm

 

 

 

 

 

ooom oooe >(Wooom oooa com o
_ ,1 _ _ _ _ _
l.o
\‘
mu.
\ \
m Illa \D .
3 o \ x
I II bx \- 18
ma I \\ s
\ \o
:55 nummo mcflucmam \\ \. %
I I es
ov n
3
G
T...
s
a
TI Inom %
9
II .llom
II llooa
_ r _
_ 2 _ 2 _

 

 

 

24

.mcofiuommcfl mo nomads may no muflmcmc Edasooca wo poommm

Hfiom m\monomm

.m musmflm

 

ooom ooov ooom oooa com o
_ IT _ é _ _ _ 5
ml \..
I. \.DI.\-\ II
C\\.\\:
\.\|\\
u
1 l

 

 

m
III. 3

 

l I 3
ES 5me @2383 I

P F P _

om

OOH

oma

oom

omm

 

 

suorqoegul zeqmnN

% Smut Disease (Probits)

25

 

 

 

 

I I I I I
99 " . '-
Planting Depth (mm)
a 15
II10
O 5
D
— D —
7 I.
0 I
— —+
II
50 ~13 . .-
I— d
0
O
lOI— _.
1 _. .4
J I I I I
250 500 1000 2000 4000

Figure 3.

Log Spores/g

The relation between percent smut disease
(probability scale) and the number of
spores/g of soil (logarithmic scale).

26

ordinate is symmetrical about the ED and either percent—

50’
age healthy or percentage diseased plants gives a linear
relation. In these experiments the slope was approximately
0.5 for the three curves which represent the three planting
depths. However at the higher inoculum densities the slope
of the curve flattened.

The data from Table l are plotted as log number of
infections versus log number of spores/g of soil (Figure 4).
The relation between these two variables on a log-log scale
was only linear over the inoculum range of 250-2,000 spores/g
of soil, where the slope ranged from 0.64 to 0.72, and the
correlation coefficient ranged between 0.90 and 0.95. How-
ever, above 2,000 spores/g of soil the slope flattened
significantly. Thus the actual relation between the log
number of spores/g of soil and log number of infections was
very close to the predicted linear relation between inoculum
density and disease severity up to 2,000 spores/g of soil
(2, 3).

Disease incidence in response to increasing inoculum
densities, when measured as the distance between spores,
was linear (Figure 5). The correlation coefficient ranged
between 0.90 and 0.95 for the entire inoculum range tested.
A decrease in the distance between spores, which is another
way of expressing an increase in the number of spores/g of
soil, resulted in a linear increase in disease incidence.

That is, a unit change in the distance between propagules

Log Number Infections

27

 

l I l T T

— -

Planting Depth (mm)

    
    

 

 

300 - O 15 -
I 10 D
. 5
200 - _
100 _
80-
60-
40'-
20*-
10h- I.
I I I I I
250 500 1000 2000 4000

Log Spores/g

Figure 4. The relation between the log number of in-

fections and the log number of spores/g of
soil.

 

100

% Smut Disease

28

 

  
 
 

 

I I

Planting Depth (mm)
D 15

I 10
I 5

 

 

 

 

Figure 5.

2.0
Distance between PrOpagules (mm)

The relation between percent smut disease
and the calculated distance between spores.

29

produced a unit change in percent diseased plants over all
inoculum densities tested. Inoculum density measurements
have been normally given as the number of propagules/g of
soil, or conidia/ml (2, 11); however, a more meaningful way
of expressing this measurement in soil may be the distance

between propagules.

Effect of temperature

 

Eight inch plastic pots in water bath controlled
temperature tanks at 12, 16, 20, 24, and 28°C in an evap-
oratively cooled greenhouse were used to measure the effect
of temperature on the relation of inoculum density to
disease incidence. Disease incidence rose with increasing
temperature, reaching a maximum at 20°C, and decreased at
24 and 28°C (Figure 6). At 12°C the amount of infection
after five weeks was near zero, however when plants were
removed from 12°C and incubated for two weeks at 20°C, the
disease incidence rose approximately 10%.

The effect of temperature on percent diseased plants
was analogous to the effect of temperature on spore germin-
ation $2.2iE52 (23), although the optimum temperature for
spore germination in_yi3£2_was somewhat higher.

There was a significant difference in the disease
response at 16 and 20°C as inoculum was increased from
1,000 to 2,000 spores/g of soil. Two thousand spores/g of

soil gave 49% infection at 16°C and 53% at 20°C, while

% Smut Disease

30

 

 

 

 

I I T I
Spores/g Soil
50 ——— 2000
1000
,I
I
40
20
0
l I I l

 

 

 

12 16 20 24 28
Temperature (C)

Figure 6. The effect of temperature on disease incidence
at two inoculum levels.

31

1,000 spores/g of soil gave 27% infection at 16°C and 48%
at 20°C. There was a much greater decrease in percent
diseased plants at 16°C at the lower inoculum density than

at the higher inoculum density.

Effect of planting depth

 

Onion seed germination is characterized by cotyledon
elongation, most of which occurs underground. Most, if not
all, smut infection occurs in the cotyledon (1). It was
assumed that increased planting depth directly increased
the amount of cotyledon elongation in soil, and thereby
increased the amount of susceptible host tissue exposed to
inoculum. Deeper planting increased time of exposure to
inoculum slightly (Table 3), but the effect was small and
could not be separated from increased exposure of host
tissue. The amount of host tissue surface area exposed at
the 5 mm planting depth was taken as one unit, the amount
exposed at the 10 mm planting depth as two units, and the
amount exposed at 15 mm planting depth as three units.
Percentage diseased plants at four inoculum densities and
three planting depths was determined (Table l), and plotted
as the relative amount of susceptible host surface area
exposed to inoculum (Figure 7). Disease incidence was
directly prOportional to the amount of susceptible host
tissue exposed to inoculum at all inoculum levels tested.

Percentage diseased plants, for example, at 250 spores/g

% Smut Disease

32

 

 
   
    
  
 
  

I l I
90 h Spores/g Soil -
A 2000
D 1000
P I 500 I
O 250
70I- '—
50 L. .—
30 _. .—
10 -' '-
l I I

 

 

 

w

1 2
Host Surface Area

Figure 7. The effect of relative host surface area
exposed to four inoculum densities on per-
cent disease.

33

increased from 15 at one unit of host surface area to 29%
at two units, and to 46% at three units. Percent diseased
plants at 2,000 spores/g increased from 49 at one unit, to
67% at two units, and to 89% at three units of host surface
area. At all inoculum densities, the average increase in
diseased plants with increased exposure of host tissue due
to deeper planting was approximately 20%.

The calculated number of infections resulting from
seeds planted 10 mm deep was approximately twice that of
seeds planted at 5 mm (Table 1). However, increasing the
planting depth from 10 to 15 mm again doubled the number of
infections, which was twice the increase expected from
extrapolation of the difference between 5 and 10 mm planting
depth. For example, with 500 spores/g, 48 infections
occurred at 10 mm planting depth and 94 infections at the
15 mm planting depth, a doubling of the number of infec-
tions. At 2,000 spores/g, 100 infections occurred at 10 mm
compared to 210 at the 15 mm planting depth. The number of
infections resulting from seeds planted 15 mm deep was thus
approximately twice that of seeds planted 10 mm deep. The
doubling in disease severity at the 10 mm planting depth
over the 5 mm depth was expected because twice as much host
tissue was exposed to inoculum. However, the increase in
infections at the 15 mm depth cannot be explained by in-
creased surface area alone, since surface area was not

doubled.

34

Effect of inoculum placement

 

The effect of planting depth on disease incidence
raised the question of whether all parts of the cotyledon
are equally susceptible, and whether the response of the
cotyledon is the same or different when exposed to a given
amount of inoculum at different planting depths. That is,
does the additional expenditure of energy required for
emergence from deeper planting result in a higher degree of
susceptibility and more infection from a constant amount of
inoculum? It was known from preliminary experiments that
inoculum placed below the seed was not effective in produc-
ing disease, even if the seed was placed just above the
inoculum.

Seeds were planted 5, 10, and 15 mm below the soil
surface in these experiments (Table 2). Infested soil
(1,000 spores/g) was placed in the following zones: (a)
the t0p 5 mm, (b) 5 to 10 mm below the surface, (c) 10 to
15 mm below the surface, (d) the upper 10 mm, (e) from 5 to
15 mm below the soil surface, and (f) the entire 15 mm.
More disease occurred from inoculum in the 5 mm zone imme-
diately above the seed than occurred from a 5 mm inoculum
zone separated from the seed by 5 mm of uninfested soil.
Sixty-seven percent diseased plants occurred when spores
were confined to the first 5 mm above seeds planted 15 mm
below the soil surface. Eighty percent diseased plants

occurred when spores were present in all three 5 mm zones

35

Table 2.--The effects of planting depth and infestation of

soil by zones on percent disease

 

 

 

Seed Percent Smut Disease
Placement (Location of infested soila zone mm)
depth mm 0 - 5 5 - 10 10 - 15 0 - 10 5 - 15 0 - 15
5 9.0 3.1 0.5 19.3 4.9 20.1
10 16.9 25.2 3.4 47.8 22.3 49.6
15

18.2 47.5 67.0 61.4 70.9 79.5

 

aSoil infested at 1,000 spores/g

36

of soil covering the seeds. Also, when seeds were planted
10 mm deep and infested soil was used in all three 5 mm
zones, 50% disease occurred, compared with 25% when inoculum
was confined to the 5 mm immediately above the seeds.

The amount of disease from inoculum present in the
zone 5 to 10 mm below the soil surface was greater when
seeds were planted 15 mm deep than when seeds were planted
10 mm deep. Also, more infection occurred from inoculum
present in the t0p two 5 mm zones of soil when seeds were
planted 15 mm deep than when seeds were planted 10 mm deep.
It appeared that more disease occurred from inoculum in
soil in close proximity to the seed than occurred from

inoculum in soil above this region.

Time of exposure to inoculum

In the temperature and planting depth studies the
time of exposure of susceptible host tissue to inoculum
also varied because lower temperatures and deeper planting
depths delayed seedling emergence. The length of exposure
is an important variable in the disease response (3). Since
it was not possible to separate length of exposure to inoc—
ulum from direct effects of temperature or depth, measure-
ments of time required for emergence of the cotyledon and
appearance of the first true leaf are reported (Table 3).
Cotyledons emerged from soil in seven days when planted 5

mm below the soil surface. Cotyledon emergence was delayed

37

Table 3.--Number of days after planting required for 50%
emergence of cotyledons and 50% appearance of
true leaves

 

 

 

 

Planting depth Temperature Days required for 50% of:
Cotyledons First leaves
5 mm 20°C 7 18
10 mm 12°C 10 23
16°C 8 21
20°C 8 l9
24°C 6 16
28°C 4 15

15 mm 20°C 9 19

 

38

by one day at the 10 mm planting depth and by two days at
15 mm planting depth. In addition deeper planting delayed
the appearance of the first true leaves by one day.

The effect of temperature on time of cotyledon and
leaf emergence was investigated (Table 3). At 20°C eight
days were required for cotyledon emergence and 19 days for
appearance of the first true leaves. Cotyledon emergence
was delayed by two days and true leaf appearance by four
days, at 12°C. Cotyledon emergence was not delayed at 16°C
but true leaf appearance required two more days than at
20°C. At 28 and 24°C, cotyledon emergence occurred four
and two days earlier and the first true leaves appeared

four and three days earlier than at 20°C.

Onion seedlingexudation

Since spore germination in soil is thought to occur
in response to nutrients (25, 38), experiments were designed
to determine the nutrient exudation pattern of developing
onion seeds. Four 9 onion seeds in a Buchner funnel covered
by a glass fiber filter paper were washed with 100 ml dis-
tilled water daily. The conductivity of the solution con-
taining the exudate was determined with an electrode and a
conductivity bridge. Conductivity was used as an indication
of the amount of exudation occurring. Most of the electro-
lytes were lost in two days (Figure 8). After the second
day a very small amount of electrolytes were present in the

solution containing exudate.

u Mhos/Seed

39

 

 

 

 

 

I I l l I I
l 3 4 5 6 7
Days
Figure 8. Conductivity of exudates of onion

seeds germinating under moist glass
fiber filter paper and leached daily.

40

Most of the ninhydrin- and anthrone-positive mater-
ials were exuded in the first two days of incubation, sim-
ilar to the conductivity measurements (Figure 9). Seeds
were treated with Clorox in an attempt to reduce bacterial
contamination; however, after one day the number of bacteria
present in the exudate from Clorox-treated seeds was not
significantly lower than that in untreated seeds. Ninety
percent of the untreated seeds germinated in 48 hours, but
Clorox-treated seeds required 72 hours for the same level
of germination. After two days untreated seeds exuded 83%
of the total ninhydrin- and anthrone-positive materials
exuded during the first four days. After two days Clorox-
treated seeds exuded 60% of the total ninhydrin- and anthrone-
positive materials exuded during the first four days. In
either treated or untreated seeds after three days 92-95%
of the total ninhydrin- and anthrone-positive material
exuded in the first four days was recorded. Apparently
more ninhydrin- and anthrone-positive material was exuded
from the seeds during the early germination stage. The
amounts measured may be lower than the actual amounts exuded,
because some material may have been metabolized by bacteria
before collection.

Since germination and growth of the fungus (12, 23,
47) and host (46, 47) were affected by temperature, this
study was done at 12, 16, 20, 24, and 28°C. Surface-disin-

fested onion seeds were aseptically removed from nutrient

41

pees/queIEArnba euronts 5n

H
I
O

N
I
O

m
o
o

V
I
O

m.o

.MHHmc tonnes
can Momma kuawm Hmnflm mmmaw “mace nova: mcflumcHEHmm
mummm coaco an cmcdxm mowom ocwEm can mmumucmsonnmu

.m musmflm

 

 

 

 

mama
v m N H v m . N I
_ _ _ I _ _ fi 1
I xouoHo I I
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_I _ F, I _ _ _ _

 

1
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peas/quatenrnba ascents 5n

42

agar after four days, placed in a sterile container, and
covered with sterile sand. These seeds were allowed to
continue germination, and ninhydrin and anthrone determina-
tions were made daily from the sterile exudate. The process
of surface disinfestation and assay for uncontaminated seeds
required a minimum of four days. Thus all seeds selected
for this study had begun germination.

The amounts of anthrone- and ninhydrin-positive
materials in exudates from seedlings grown at temperatures
of 16, 20, and 24°C after one day were approximately 4.8
and 2.2 ug/seed respectively at all three temperatures
(Figures 10 and 11). These amounts dropped over the next
two days to 1.0 and 0.9 ug/seed respectively on the fourth
day. The amounts exuded from plants grown at 12°C were
lower, and at 28°C a significantly larger amount was exuded
for the first five days. The amounts exuded after the sixth
day at any temperature were less than 1 ug/seed. Apparently
exudation was affected slightly as temperature increased,
but at 28°C a significantly larger amount of nutrient was

exuded.

Spore germination

Spore germination in soil is thought to occur in
response to nutrients lost by host plants (25, 38). Telio-
spores which floated on the surface of water containing

10-20 germinating onion seeds all germinated; however, the

43

 

 

 

 

 

15 - q C —
\ -—-—— 28
I \ ._
_" \
\ —_ 16,20,24
_. \\ -—
\ -—-— 12
"— I .\ ""I
g _ \.. __
:I \
'gloL. \\ —
g t
\ _ \ _
‘é \
g s
.,..| \\
*- x -
m __ \. _.
U)
o
8
F, 5- .—
0
U) I.— .—
:1.
0 '_ -—-I
I I I I I I
X 2 3 4 5 6
Figure 10. Carbohydrates exuded by uncontaminated

onion seedlings after seeds were in-
cubated four days (X) on agar and
then placed in sterile chambers and
washed daily.

OJ

pg Glycine Equivalent/Seedling
N

H

44

 

 

I I I I I
\ C
‘\ -"- 28
\ — 16,20,24

 

 

 

I-— —
p. .—
I-I—n —
L I I I I I
X 2 3 4 5
Days
Figure 11. Amino acids exuded by uncontaminated

onion seedlings after seeds were in-
cubated four days (X) on agar and
then placed in sterile chambers and
washed daily.

45

majority of the spores, which sank below the surface, failed
to germinate. Later studies indicated that spores failed to
germinate when an agar surface contained a continuous water
film. This confirmed a requirement for oxygen for spore
germination, as reported by Anderson (1).

Since the seed is the only nutrient source in the
early development of the onion plant, it may also be the
major source of nutrients for teliospore germination.
Dormant onion seeds were broken and mixed in agar contain-
ing basal salts. After eight days teliospore germination
of 48, 56, and 65% occurred on agar containing 2.5, 5.0,
and 8.0 g/liter of ground onion seed respectively (Table 4).
Teliospore germination on malt extract-phytone agar (MEPA)
control was 78%. Teliospores on water agar placed over
intact, ungerminated seeds supported 58% germination.
However, if seeds were allowed to germinate for 48 hours,
rinsed, and then placed under water agar only 29% germina-
tion resulted. Teliospores germinated on water agar placed
on top of MEPA as well as when placed directly on MEPA,
presumably because nutrients diffused into the water agar
(21).

Teliospore germination on agar containing exudate
from onion seeds which were incubated for 48 hours was 45%
when the exudate was filtered and mixed with hot (60°C)
agar plus basal salts (Table 4). If the exudate was auto-

claved before mixing with agar, spore germination was only

46

Table 4.--Germination of Urocystis colchici teliospores on
agar media and on water agar placed over onion
seeds or agar media

 

 

 

 

Nutrient Source Concentration Germinationa
g/liter Percent
1. Ground onion seed 2.5 48
2. Ground onion seed 5.0 56
3. Ground onion seed 8.0 65

4. Malt extract-phytone
(MEPA) 10 & 5 78

5. Intact, ungerminated b
onion seeds, autoclaved 58

6. Intact, 24 hr. germinated
onion seeds, autoclavedb 55

7. Intact, 48 hr. germinated
onion seeds, autoclavedb 29

8. Intact, ungerminated,
surface disinfested onion
seedsb 59

9. Autoclaved exudate from
seeds allowed to germinate
48 hr. 16 2

10. Filtered exudate from
seeds allowed to germinate

48 hr. 16 45
ll. MEPA under water agar 10 & 5 78
12. Water agar l

 

aAverage number of spores germinated/100 on each of
four plates.

bTwenty seeds/plate.

47

2%. The pH of the exudate ranged from 6.8-7.4. The reason
for the loss of ability to support germination is not known.

The standard teliospore germination procedure
develOped in this laboratory (23), required that spores be
stored for 24 hours at high concentration before plating.
Spores from a single collection were plated on MEPA concen-
trated or dilute storage, with and without leaching. Spore
leaching was done in the model system described by K0 and
Lockwood (23). Teliospores were placed on sterile Milli-
pore filters on sterile sand in a petri dish. Sterile
distilled water from a separatory funnel was dripped onto
the sand at the rate of 10-30 ml/hr. The water moved
through the sand and drained from the outlet. The spores
remained sterile during the leaching process. The germina-
tion rate and level were lower after 48 hours leaching
(Table 5). After 48 hours dilute storage, germination was
10% lower than after 24 hours dilute storage. The rate of
germination was lower in treatments 1, 2, and 4, but the
level after eight days was not lower than the control
(treatment 3). Storage of spores on Millipore filters over
natural soil for 24 or 48 hours and then placed on MEPA
resulted in germination levels of 75% and 40% respectively.
It thus appears that any treatment which removes nutrients
from the teliospores tends to reduce germination.

Mycelial growth of g. colchici was reported as

excellent on a liquid medium containing sucrose (30 g/l)

48

Table 5.--Germination of Urocystis colchici teliospores on
malt extract-phytone agar at 22°C after different

 

 

 

 

treatments

Treatment Percent Germinationa

(Days of incubation)
3 4 5 6 7 8
1. plated immediately 20 32 45 56 65 79
2. 24 hr., dilute storageb 19 33 48 56 68 77
3. 24 hr., conc. storagec 22 50 68 71 76 79
4. 24 hr., leached sporesd 20 35 so 65 73 78
5. 48 hr., dilute storage 20 28 50 56 61 67
6. 48 hr., conc. storage 23 52 68 69 73 78
7. 48 hr., leached spores 18 25 30 34 43 45

 

aAverage number of spores/100 germinated on each of
four plates.

bDilute storage (103 spores/ml).

CConcentrated storage (105 - 106

dLeached spores were subjected to dripping water
for the time indicated.

spores/ml).

49

and asparagine (4.7 g/l) (12). To determine if nutrient
requirements for growth were the same as for spore germina-
tion, 16 different combinations of sucrose and asparagine
were tested for teliospore germination (Table 6). Germina-
tion ranged from 6—15%. The sucrose-asparagine medium
failed to support complete germination, but no indication

of the reason for this was obtained.

50

.mmpmHm HDOM mo some so pmumcflEHom ooa\mwuomm mo Hones: wmmum>¢m

 

 

me o.m maoumam OH uomnuxm paws .AH
ca o.m mcflmmnmmmd: . om mmouosm .ma
m o.m wcwmmummmm, om mmouosm .ma
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0H o.m mcwmmummmfl om mmouosm .HH
5 o.a ocwmmummmfi om omouosm .oa
ea m.o ocwmmummmd om mmouozm .m
NH o.m mcflmmummmd oH omouosm .m
ma o.m ocflmmummmd ca mmouosm .5
ma o.H mcwmmummmd oa mmouosm .m
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m o.m ocflmmnmmmd m mmonosm .v
ma o.m mcfimmnmmmfi m mmouosm .m
m o.H mcflmmnmmmfl m mmouosm .m
0H m.o mcflmmummmfi m omOHUSm .H
Hopwa\m Hmufia\m
coflumcfleumm GOHDMHDGGUGOU ucmflnusz coaumnucmocoo ucmfluusz

M

 

 

.IIIIIIII oomm pm mates Heme
ca mcflmmummmm Use mmouodm co monommOmeu Huanoaoo mflumNoona mo GOADMCHEHmeI.m magma

DISCUSSION

Initially only the effect of inoculum density was
considered in relating inoculum potential and disease
potential to disease severity. Theoretically the addition
of more inoculum to soil should have resulted in propor-
tional increases in disease severity: however, a point was
reached at which increased amounts of inoculum did not
increase percent disease proportionately (Figure 1). Since
disease measured by percentage ignores any increase in
disease severity that occurs on diseased plants, the
multiple-infection transformation was used to calculate the
number of infection per plant (17). The multiple-infection
transformation (Figure 2) straightened the dosage-response
curve somewhat, but response was not completely linear.

The log-probit grid (Figure 3) produced a linear response,
but only for inoculum densities below 2,000 spores/g of
soil. The log-log plot of the number of infections against
the number of spores/g of soil (Figure 4) also was linear
up to 2,000 spores/g of soil: however, above this inoculum
level the curve flattened. This decrease in the lepe of
the curve has been attributed to the lack of susceptible

tissue in which infection can occur (29, 49).

51

52

In this study a linear relationship over the inoc-
ulum range of 125-8,000 spores/g was obtained when percent
infection was plotted against the distance in mm between
spores (Figure 5). In Baker's model (page 9) the number
of propagules that fall on a surface area (rhizoplane) is
directly related to the distance between propagules (3).
Likewise the number of propagules that fall within a spe-
cific volume is also directly related to the distance be-
tween propagules (3). Since the number of propagules in a
position to infect is directly related to the distance
between the propagules, disease severity is linearly pro-
portional to the distance between propagules.

Baker and McClintock (4) first suggested that the
number of propagules/unit volume of soil and the distance
between propagules are not linearly related (Figure 12).
Since the probability of infection is directly related to
the distance between propagules and not the number of
propagules/unit volume, it becomes clear why disease re-
sponse curves flatten out at higher inoculum densities
(Figures 1 and 2). The fact that the magnitude of decrease
in distance between pr0pagules is very small as inoculum
density increases above 1,000 spores/g means that the
increased probability of further infection is also very
small. This factor has been omitted as a reason for the
typical decrease in slope of the disease response curve at

high inoculum densities, although Baker and McClintock (4)

Log Spores/g

53

 

 

 

 

 

 

 

I T I I ‘ I
._ 2000
8000 \ Spores/g -1
\ -—- Log Spores/g
4000 —
2000 —
1000 r—
500 — — 1000
250“-
- 500
100I——
- 250
__ 125
30 I—‘ _. 0
l I J I I
1 2 3 4 5
D(mm)

Figure 12. The relation between calculated distance
(D) between spores in soil and number of
spores or log number of spores/g of soil.

Spores /g

54

first suggested that this factor could be important in
limiting the density of natural pathogen populations in
soil.

Transformations using the log number of propagules/g
of soil when relating disease and inoculum densities below
1,000 propagules/g of soil are linear because the relation
between the log number of pr0pagules/g of soil and distance
between propagules is linear in this range (Figure 12).
However, above 1,000 propagules/g the relation is not linear
and the disease response curve flattens when plotted against
the log number of propagules/g of soil. This may explain
the common, so-called logarithmic effect in natural phenom-
ena, and the linearity of curves using logarithmic trans-
formations.

The position of a disease response curve with re—
spect to inoculum density may change if: (1) spore germina-
tion was less than 100%; (2) if spore viability was dif-
ferent from that assumed; (3) if more than one spore were
required for infection; or (4) if host resistance was
different: however, the curve would remain linear. A change
in position was illustrated by the planting depth exper-
iment (Figure 7).

The response to inoculum present in the top 5 mm of
soil was not increased by planting deeper: however, if
inoculum was placed in the zone 5 mm above the seed a sig-

nificant increase in infection occurred as planting depth

55

was increased (Table 2). The length of exposure to inoculum
contained in the top 5 mm of soil was not increased by
deeper planting. However, the length of exposure to
inoculum in the 5 mm zone immediately above the seed was
increased by deeper planting. This increase in disease is
probably due to increased time of exposure of susceptible
tissue to inoculum placed in the immediate vicinity of the
seed. It appears that the cotyledon is uniformly suscep-
tible and that the increase in disease at deeper plantings
was due to increased time of exposure.

The primary soil factors influential in disease
incited by soil-borne pathogens are: temperature, moisture,
soil type, pH, and fertility (44). The effect of temper-
ature on the relation between inoculum density and disease
was best shown at 16°C. The difference in amounts of
disease with 1,000 or 2,000 spores/g may be explained, in
part, by the effect of temperature on spore germination in
yitgg. Spore germination data (23) indicate 48% germination
at 16°C and 84% at 24°C after eight days. Assuming that
approximately half of the spores in soil would germinate at
16°C that would germinate at the optimum temperature, as
was shown in yitgg (23) the reduction in disease at 16°C
might have been predicted, because the effective inoculum
level was reduced by nearly half. Thus 2,000 spores were
reduced to 1,000 effective spores/g and 1,000 to 500 effec-

tive spores/g because of incomplete germination at the

56

below-Optimum temperature. The temperature effect on rate
of emergence (Table 3) and fungus growth at 16 and 20°C was
reported to be small (12, 47). The change in response of a
population of plants to a decrease in inoculum density from
2,000 to 1,000 spores/g was very small (Figure 5); however,
the change in response to a decrease from 1,000 to 500
spores/g was much greater. In the past, the effect of sub-
optimal temperature on amount of disease may have been
masked by using a high inoculum level (46).

Although the effect of temperature on amount of
disease at 16°C may be explained by a reduction of spore
germination, other factors may become important at 12, 24,
and 28°C. At 12°C the length of exposure of host tissue to
inoculum was increased (Table 3), but germination and growth
of the fungus were reported to be very low (12, 23): there-
fore amount of disease would be limited (Figure 7). Spore
germination and vegetative growth of the fungus were re-
ported to be optimum at 24°C (12, 23), but were nearly as
good at 20°C where optimum infection occurred. The mean
length of exposure of the cotyledon to inoculum was six
days at 24°C and eight days at 20°C. The first true leaf
appearance was three days earlier at 24°C than at 20°C.

The appearance of the first true leaf was stated to be
approximately coincident with the development of immunity
in the onion cotyledon (l, 46); thus, at 24°C immunity to

infection may have been reached earlier. At 28°C the length

57

of exposure of host tissue to inoculum was shortest and the
immune condition occurred earliest as judged by the appear-
ance of the first leaf. Also the growth and germination of
the fungus were reported to be restricted at 28°C (12, 23,
47). Therefore, even when exudation was greatest, the
amount of disease was low. There was general agreement,
with results reported earlier (46), that infection can
occur at either high or low temperature, but that at above
25°C a decided reduction in disease occurs.

Germinating onion seeds and developing onion
cotyledons were demonstrated to exude soluble organic
nutrients. The amount of material exuded was not abundant,
but present in sufficient quantity to support teliospore
germination. Seeds with young emerging radicles were the
major source of nutrients. This early exudation pattern
was also suggested from inoculum studies where infested
soil was placed in zones. The amount of disease was always
greatest when inoculum was placed in the zone in which
germination began. However the zone in which germination
began always was exposed to inoculum for a longer time.

Exudation at temperatures of 12, 16, 20, and 24°C
was not significantly different, but the amount of material
exuded at 28°C was higher than the amounts recovered at 12-
24°C. Increased exudation at 28°C would not affect smut
disease severity because both germination and growth of the

fungus were limited at this temperature (12, 23, 47).

58

Onion seedlings also develop rapidly at 28°C, shortening
the time of exposure to inoculum. The amounts of ninhydrin-
and anthrone-positive materials exuded from sterile onion
seedlings were much higher than the amounts recovered from
nonsterile seedlings. This may have been due to: (a)
lowering of amounts of recoverable materials by contaminat-
ing microorganisms, (b) absorption of nutrients from agar
by the seed and emerging radicle which were later recovered
in exudates, or (c) increased release of nutrients due to
the shock of transplanting into the sand system.

Although no direct evidence that specific substances
were exuded to selectively cause pathogen spores to ger-
minate, onion seeds and exudates from germinating onion
seeds were shown to be sufficient to support teliospore

germination, which could lead to successful infection.

LITERATURE CITED

10.

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